Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium-activated K+ channels are voltage- independent and share a common Ca2+/calmodulin mediated gating mechanism. Their lack of voltage- dependence enables KCa2/3 channels to remain open at negative membrane potentials and the channels therefore play important roles in physiological processes that require hyperpolarization. While the three KCa2 channels, KCa2.1 (SK1), KCa2.2 (SK2) and KCa2.3 (SK3) are best known for their role in neuronal afterhyperpolarization, KCa3.1 (IK) has mostly been studied in the immune system, vascular endothelium and in secretory epithelia, where the channel is involved in activation, proliferation and secretion processes through modulation of Ca2+ influx events. Small molecule KCa2/3 channel modulators constitute both useful chemical biology probes as well as potential novel drugs for the treatment of autoimmune diseases, hypertension, and various neurological disorders such as ataxia, epilepsy, and alcohol dependence. Our laboratory has been working on the pharmacology of KCa2/3 channels for many years. After we initially developed KCa3.1 blockers such as TRAM-34, we later discovered the mixed KCa2/3 activator SKA-31 and the KCa3.1 selective activators SKA-121 and SKA-111, which display 40- or 100-fold selectivity for KCa3.1 over KCa2 channels. All these compounds, which have been widely used to probe the physiological and pathophysiological functions of KCa channels, were designed using classical medicinal chemistry approaches without any structural input. However, using the recently solved crystal structures of the KCa2.2 calmodulin binding domain (CaM-BD) in complex with CaM from our consultant Miao Zhang, we generated Rosetta homology models of the KCa2.3 and KCa3.1 CaM-BD/CaM complexes and discovered that an extensive hydrogen bond network stabilizing SKA-121 in KCa3.1 is key to its KCa3.1 selectivity. Using this atomistic scale structural insight into KCa channel subtype selectivity we are now proposing to switch selectivity under Aim-1 and perform hypothesis-driven structure-assisted drug design of novel napthothiazole/oxazole-type KCa activators that make unique contacts with KCa2-specific residues using the Rosetta Ligand and the new RosettaDrug Design approach. After synthesizing and experimentally testing KCa channel potency and selectivity by electrophysiology, we intend to first confirm the binding mode by mutagenesis and then turn the new KCa2 activators into a useful pharmacological probe for the scientific community under Aim-2, where we will determine selectivity over other ion channels and evaluate pharmacokinetic properties and brain penetration. The innovation in our proposal is twofold: 1) This work will be one of the first attempts at hypothesis-driven structure based drug design for a small molecule ion channel modulator; 2) This work will provide the scientific community with KCa2 channel selective gating modulators which will be useful tools to explore the pathophysiological role of KCa2 channels and their suitability as therapeutic targets for epilepsy, ataxia, substance dependence and post-traumatic stress disorder.